Method of analysis, reagent composition and use thereof for glucose determination

Abstract

A method, a reagent composition and the use thereof for quantitative determination of total glucose in undiluted whole blood are described. A sample of undiluted whole blood is contacted with a reagent composition which is in dry form and comprises: glucose dehydrogenase (GDH); one or more substances from the group consisting of diaphorase, phenazine methosulphate, phenazine ethosulphate, phenazine phenosulphate and Meldola blue; one or more substances from the group consisting of NAD, NADP, thio-NAD, thio-NADP, nicotinamide-purine dinucleotide, nicotinamide-methylpurine dinucleotide, nicotinamide-2-chloro-methylpurine dinucleotide; one or more hemolysing substances from the group consisting of phospholipase, hemolysing saponins, and compounds of hydrophilic mono-, di- or tri-saccharides and aliphatic hydrocarbons having 10-16 carbon atoms; a redox indicator dye; and optionally mutarotase. A color change brought about by the reaction of the reagent with glucose in the undiluted whole blood is measured by transmission spectrophotometry.

We claim: 
     
       1. A method to quantitatively determine the glucose concentration of a sample of undiluted whole blood, comprising contacting a sample of undiluted whole blood with a reagent in dry form in a microcuvette to obtain hemolyzed undiluted whole blood, the reagent comprising: glucose dehydrogenase (GDH);   one or more substances from the group consisting of diaphorase, phenazine methosulphate, phenazine ethosulphate, phenazine phenosulphate and Meldola blue;   one or more substances from the group consisting of NAD, NADP, thio-NAD, thio-NADP, nicotinamide-purine dinucleotide, nicotinamide-methylpurine dinucleo-tide and nicotinamide-2-chloro-methylpurine dinucleo-tide;   one or more hemolyzing substances from the group consisting of phospholipase, hemolyzing saponins, compounds of hydrophilic mono-, di- or tri-saccharides and aliphatic hydrocarbons having 10-16 carbon atoms; and   a redox indicator dye; and measuring by transmission spectrophotometry a color change brought about by the reaction of the reagent with glucose in the undiluted whole blood to quantitatively determine the glucose concentration of undiluted whole blood.     
     
     
       2. The method as claimed in claim 1, wherein the sample of undiluted whole blood is a volume of 0.1 to 0.001 ml. 
     
     
       3. A reagent composition to quantitatively determine the total glucose concentration of undiluted whole blood, wherein the reagent composition is in dry form and the reagent composition comprises: glucose dehydrogenase (GDH);   one or more substances form the group consisting of diaphorase, phenazine methosulphate, phenazine, ethosulphate, phenazine phenosulphate and Meldola blue;   one or more substances from the group consisting of NAD, NADP, thio-NAD, thio-NAD, nicotinamide-purine dinucleotide and nicotinamide methylpurine dinucleotide;   one or more hemolyzing substances from the group consisting of phospholipase, hemolyzing saponins, saccharides and aliphatic hydrocarbons having 10-16 carbon atoms; and   a redox indicator dye.   
     
     
       4. The reagent composition as claimed in claim 3, wherein the redox indicator dye is a tetrazolium salt. 
     
     
       5. The reagent composition as claimed in claim 3 wherein the reagent composition comprises: glucose dehydrogenase,   diaphorase,   NAD,   one or more saponins, and   a tetrazolium salt.   
     
     
       6. A reagent composition used to quantitatively determine the glucose concentration of a sample of undiluted whole blood, the reagent composition comprising: glucose dehydrogenase (GDH);   one or more substances from the group consisting of diaphroase, phenazine methosulphate, phenazine ethosulphate, phenazine phenosulphate and Meldola blue;   one or more substances from the group consisting of NAD, NADP, thio-NAD, thio-NADP, nicotinamide-purine dinucleotide, nicotinamide-methylpurine dinucleotide and nicotinamide-2-chloro-methylpurine dinucleotide;   one or more hemolyzing substances from the group consisting of phospholipase, hemolyzing saponins, compounds of hydrophilic mono-, di- or tri-saccharides and aliphatic hydrocarbons having 10-16 carbon atoms; and   a redox indicator dye; and which composition is in dry form in a microcuvette, for carrying out quantitative determination of total glucose in undiluted whole blood by transmission spectrophotometry to quantitatively determine the glucose concentration of undiluted whole blood.     
     
     
       7. The method as set forth in claim 1 further comprising the step of introducing and freeze drying the reagent in a microcuvette prior to said contacting. 
     
     
       8. The reagent composition of claim 3 wherein said reagent composition is freeze dried in a microcuvette. 
     
     
       9. The method of claim 1 wherein the reagent further comprises mutarotase. 
     
     
       10. The reagent composition as set forth in claim 3 wherein the reagent composition further comprises mutarotase. 
     
     
       11. The reagent composition as set forth in claim 5 wherein the reagent composition further comprises mutarotase. 
     
     
       12. The reagent composition as set forth in claim 6 wherein the reagent composition further comprises mutarotase.